The present invention relates to a nucleic acid construct harboring a constitutive non-tissue specific promoter for the higher expression of the desired polypeptide in plant cells and utilization thereof, particularly to a promoter for genes capable of being constitutively expressed in the highest level in transgenic plant root tissues and leaf tissues. The gene expression by said construct is much stronger than that of 35S promoter. The invention is exemplified by the use of Arabidopsis TPS3 promoter for the constitutive regulation of gene expression to produce desirably transformed plants.
For carrying out the crossing, the conventional breeding is problematic in that the genotype to be introduced is limited to one for relative crossing and that it takes a long period of time to obtain the intended hybrids. Using to recent biotechnological development such as genetic engineering technology, however, a desirable gene can be directly introduced into plants, and such system is expected to be capable of overcoming the problems in the conventional breeding. According to the plant genetic engineering, it is also very advantageous in producing plants with improved traits. For improving the resistance to plant diseases, insects and environmental stresses or enhancing the nutritional characteristics of transgenic plants, a strong constitutive promoter which controls expression of the gene should be linked to the desired gene, where it makes the gene be expressed under the control of said promoter. Also, the economical production of biologically active polypeptides is important for the manufacture of pharmaceutical and nutritional formulations and other specialty chemicals in plant biofarming. Recombinant DNA techniques, using transgenic plant cell as expression host generated with a recombinant nucleic acid construct harboring strong constitutive promoter, are particularly useful means for producing large quantities of polypeptides.
Several promoters useful in expressing heterologous genes in plants have already been identified. The most widely used promoter is the 35S promoter from the cauliflower mosaic virus (CaMV35S promoter). The 35S promoter is a strong, constitutive promoter that causes gene expression in all tissues of transgenic plant. Other constitutive promoters that have also been identified include those from the mannopine synthase gene and the nopaline synthase gene of the T-DNA of Agrobacterium tumefaciens. For producing recombinant polypeptides that are intended for commercial use, in particular, it is required to obtain a high level of expression of the desired polypeptide from the host cells. Therefore, it requires an expression control signal that is capable of conferring the highest level of strong constitutive expression of the desired polypeptide in plant tissues that maintains expression characteristics. Access to such constitutive promoter would enable the genetic engineering of commercially important crops. The present invention completed this.
Regarding Arabidopsis TPS3 gene, the complete sequence of cDNA of Arabidopsis, which comprises 2,583 bases, has been reported, and the amino acid sequence thereof comprising 861 residues has also been deduced (see GenBank AC004473). However, regarding the promoter region that acts to control the transcription of said TPS3 gene, there is currently no report and/or invention referring to the identification of Arabidopsis TPS3 promoter that causes very strong, constitutive gene expression of a desired gene and maintains expression characteristics in root- and leaf-tissues in transgenic plants.
According to the present invention, therefore, a desired foreign gene and a terminator can be linked to the downstream site of Arabidopsis TPS3 promoter region obtained, and introduced into plant, whereby the desired gene can be expressed in the transgenic plant tissues. Thus, the promoter region can be utilized to afford biotic or abiotic stress-related transcription and expression of a desired gene for the improvement of plants and also to produce the biologically active substances in plant tissues.
The present invention provides isolated nucleic acid constructs that comprise a plant promoter operably linked to a heterologous gene for the highest expression level of a desired polypeptide in plant root tissue and leaf tissue. The plant promoter comprises stronger constitutive promoter, from Arabidopsis gene that encodes trehalose-6-phosphate synthase involved in trehalose metabolism, than CaMV 35S promoter. Also provided by the invention are expression vectors which include Arabidopsis TPS3 promoter operably linked to a heterologous gene that encodes a desired polypeptide.
The expression vectors can further comprise other components such as a selectable marker. The construct can also comprise an origin of replication sequence that functions in plant or other host cell. As preferred constructs of the invention, the plasmid pLES 99010 was deposited under Budapest treaty with the Korean Collection for Type Cultures (KCTC) located at #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea with accession No. KCTC 0811BP on Jul. 1, 2000. Plasmid pLES 99011 was deposited under Budapest treaty with the Korean Collection for Type Cultures (KCTC) with accession No. KCTC 0812BP. Plasmid pLES 99014 was deposited under Budapest treaty with the Korean Collection for Type Cultures (KCTC) with accession No. KCTC 0813BP. Plasmid pLES 99015 was deposited under Budapest treaty with the Korean Collection for Type Cultures (KCTC) with accession No. KCTC 0814BP.
The present invention also provides a plant cell that contains a expression cassette that includes the Arabidopsis TPS3 promoter operably linked to a heterologous gene. The expression cassette can be integrated into the genome of the host cell or be present on an independently replicating plasmid. With said expression cassette, a gene manipulation of plants is possible, which enables the constitutive regulation of a desired gene expression in commercially important plants. A preferred plant cell is both dicot and monocot species.
The present inventors have conducted extensive researches and found a promoter capable of functioning in plant tissues, which enables very strong constitutive expression of desired genes, thereby completed the present invention.